CN111228479A

CN111228479A - anti-PD-L1 antibody preparation

Info

Publication number: CN111228479A
Application number: CN201911190037.4A
Authority: CN
Inventors: 肖亮; 邓莉萍; 黄海玉; 刘启刚; 刘立平; 郭大成; 薛彤彤; 王晶翼
Original assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date: 2018-11-29
Filing date: 2019-11-28
Publication date: 2020-06-05
Anticipated expiration: 2039-11-28
Also published as: US20230287122A1; EP3888678A1; JP2022513693A; CA3121288A1; EP3888678A4; CN111228479B; KR20210096640A; WO2020108497A1; AU2019388808A1

Abstract

The present invention relates to the field of therapeutic antibodies. In particular, the invention relates to an anti-PD-L1 antibody formulation comprising an anti-PD-L1 antibody, a buffer, sodium chloride, and the absence of added sugars and/or sugar alcohols. The anti-PD-L1 antibody formulation of the invention is capable of maintaining antibody stability after long term storage. In addition, the invention also relates to the application of the anti-PD-L1 antibody preparation in preparing a medicament for preventing and/or treating and/or assisting in treating and/or diagnosing tumors or anemia.

Description

anti-PD-L1 antibody preparation

Technical Field

The invention relates to the field of therapeutic antibodies, in particular to an anti-PD-L1 antibody preparation and medical application thereof.

Background

PD-1(Programmed Death 1) is a type I transmembrane glycoprotein and belongs to a member of the CD28 family. PD-L1(Programmed Cell Death-Ligand 1) is a type I transmembrane glycoprotein of 40kDa in size, known as Programmed Death receptor-Ligand 1.

Under normal physiological conditions, after antigen enters the body, it is processed by Antigen Presenting Cells (APC) and linked to MHC. T cell surface TCR molecule is through the antigen peptide that APC presents of specificity recognition, and discern MHC molecule forming complex with antigen peptide at the same time, in addition T cell surface numerous immunoregulation molecules participate in the reaction, produce the costimulation signal, impel T cell activation and give play to the immune effect. PD-1 is expressed on the surfaces of activated T cells, B cells, NK cells, monocytes and DC, interacts with PD-L1, PD-L2 and the like on the surface of APC cells, can transmit inhibitory signals, inhibits the activation of T cells and maintains the immune homeostasis of an organism. High-expression PD-L1 often exists on the surface of the tumor cell, and is combined with high-expression PD-1 of tumor infiltration T lymphocytes, so that a PD-1 pathway is continuously activated, the activation of the T cells is inhibited, and the immune escape of the tumor is realized. Therefore, blocking the combination of PD-1/PD-L1 becomes one of the important strategies in the field of tumor immunotherapy. The PD-L1 antibody can block the binding of tumor PD-L1 and T cell PD-1, eliminate the immunosuppressive effect, and reactivate T cells, recognize and kill tumor cells.

WO2017/148424 discloses a novel anti-PD-L1 monoclonal antibody which is very effective in blocking the binding of PD-1 to PD-L1 and shows a better therapeutic effect on tumors or anemia. However, the use of the PD-L1 antibody for clinical treatment requires solving the problems of physicochemical properties suitable for administration and stability during storage. Antibody formulations are the final form of antibodies in storage, transportation and clinical treatment, and need to be compatible with physicochemical properties, physiological properties and stability over the useful life, such as protein content, pH, osmotic pressure, insoluble particles, purity, aggregates, binding activity, etc. Because different antibodies have various characteristics in the aspect of physicochemical properties, the stability is greatly different, and a stable and universal liquid preparation formula is difficult to obtain in preparation of the preparation. Therefore, each antibody which is innovatively developed needs to be deeply researched, and a proper preparation prescription is searched to obtain an antibody preparation with good physiological compatibility, so that the storage of medicines and clinical medication are facilitated.

Therefore, it is necessary to develop a suitable formulation for the anti-PD-L1 antibody to obtain an antibody preparation satisfying the requirements for physiological compatibility and stability.

Disclosure of Invention

The present inventors have unexpectedly found, through a large number of experiments and repeated investigations, that a liquid formulation composed of an anti-PD-L1 antibody and a specific prescription has excellent physiological compatibility and stability, and further developed a high-concentration antibody formulation, and a high-concentration protein formulation overcomes the problems of poor stability, high viscosity and susceptibility to turbidity, and thus the present invention has been completed by disclosing a liquid formulation which is particularly advantageous for long-term storage and clinical administration of a pharmaceutical product.

Liquid preparation

Accordingly, in one aspect, the present invention provides a liquid formulation comprising:

(1) an anti-PD-L1 antibody at a concentration of about 10mg/ml to about 150 mg/ml; wherein the anti-PD-L1 antibody comprises heavy chain CDRs as shown in SEQ ID NOs:1-3, respectively, and light chain CDRs as shown in SEQ ID NOs:4-6, respectively;

(2) a buffer at a concentration of about 5mM to about 40 mM;

(3) sodium chloride at a concentration of about 80mM to about 160 mM;

wherein the liquid formulation has a pH of about 5.0 to about 7.0; and, the liquid formulation is absent added sugar and/or sugar alcohol;

also, liquid formulations have an osmotic pressure comparable to human blood.

The anti-PD-L1 antibody liquid formulations disclosed herein are characterized by the absence of added sugars and/or sugar alcohols and thus may also be referred to herein as sugarless antibody formulations.

In certain preferred embodiments, the liquid formulation does not contain a sugar and/or sugar alcohol selected from the group consisting of: sucrose, glucose, trehalose, mannitol and sorbitol.

In certain preferred embodiments, the anti-PD-L1 antibody has the heavy chain variable region as shown in SEQ ID NO. 7 and the light chain variable region as shown in SEQ ID NO. 8. In certain preferred embodiments, the anti-PD-L1 antibody has a heavy chain as shown in SEQ ID NO. 9 and a light chain as shown in SEQ ID NO. 10.

In another preferred embodiment, the concentration of the anti-PD-L1 antibody is 10mg/ml to 30 mg/ml.

In another preferred embodiment, the concentration of the anti-PD-L1 antibody is 10mg/ml to 120mg/ml, preferably 30mg/ml to 70 mg/ml.

In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is from about 15mg/ml to about 25 mg/ml. In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is about 20 mg/ml.

In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is from about 50mg/ml to about 70 mg/ml. In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is about 60 mg/ml.

In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is from about 80mg/ml to about 150 mg/ml. In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is about 90mg/ml, about 100mg/ml, about 110mg/ml or about 120 mg/ml.

In certain preferred embodiments, the concentration of sodium chloride is from about 100mM to about 160 mM. In certain preferred embodiments, the concentration of sodium chloride is about 105mM, about 110mM, about 115mM, about 120mM, about 130mM, about 135mM, about 140mM, about 145mM, about 150mM, or about 155 mM. In certain more preferred embodiments, the concentration of sodium chloride is about 140 mM.

In certain preferred embodiments, the buffer is selected from: citrate-citrate buffer, histidine buffer, or acetate-acetate buffer. In certain preferred embodiments, histidine is present in the histidine buffer alone or as histidine hydrochloride or histidine acetate.

In certain preferred embodiments, the buffer is a histidine buffer. In certain preferred embodiments, histidine is present in the histidine buffer alone or as histidine-hydrochloride or histidine-acetate.

In certain preferred embodiments, the buffer is at a concentration of about 10mM to about 20 mM. In certain preferred embodiments, the buffer is at a concentration of about 20 mM. In certain preferred embodiments, the liquid formulation comprises histidine buffer at a concentration of about 20mM, preferably histidine in the histidine buffer is present alone or as histidine hydrochloride or histidine acetate.

In another preferred embodiment, the buffer is 20mM histidine-histidine hydrochloride.

In the present invention, the pH of the liquid formulation is adjusted by a buffer, which may further adjust the pH using a pH adjusting agent (e.g., hydrochloric acid, acetic acid, phosphoric acid, etc.). In certain preferred embodiments, the liquid formulation has a pH of about 5.2 to about 6.8. In certain preferred embodiments, the liquid formulation has a pH of about 5.6 to about 6.8. In certain preferred embodiments, the liquid formulation has a pH of about 5.6 to about 6.0. In certain exemplary embodiments, the pH of the liquid formulation is about 5.0, about 5.2, about 5.6, about 5.8, about 6.0, about 6.4, about 6.8, or about 7.0. In certain exemplary embodiments, the pH of the liquid formulation is 5.8 ± 0.1. In certain exemplary embodiments, the pH of the liquid formulation is about 5.8.

In certain preferred embodiments, wherein the liquid formulation has an osmolality of about 260 to about 380 mOsmol/kg. In certain preferred embodiments, the liquid formulation has an osmolality of about 280 to about 380mOsmol/kg (e.g., 280 to about 360 mOsmol/kg). In certain preferred embodiments, the liquid formulation has an osmolality of about 280mOsm/kg to about 330 mOsmol/kg. In certain preferred embodiments, the liquid formulation has an osmotic pressure substantially the same as human blood.

In another preferred embodiment, the osmolality equivalent to human blood is from about 280 to about 400mOsmol/kg, preferably from about 290 to about 320mOsmol/kg, more preferably from about 295 to about 315 mOsmol/kg.

In certain preferred embodiments, the liquid formulation further comprises a surfactant. In certain preferred embodiments, the surfactant is polysorbate 80/polysorbate 20 or poloxamer 188. In certain preferred embodiments, the surfactant is polysorbate 80.

In certain preferred embodiments, the concentration of the surfactant is from about 0.01% w/v to about 0.02% w/v. In certain preferred embodiments, the concentration of the surfactant is about 0.02% w/v. In certain preferred embodiments, the liquid formulation comprises polysorbate 80 at a concentration of about 0.02% w/v.

In certain preferred embodiments, the liquid formulation comprises:

(1) the anti-PD-L1 antibody at a concentration of about 20mg/ml, about 60mg/ml, about 90mg/ml, or about 120 mg/ml;

(2) a buffer at a concentration of about 20 mM; the buffer is selected from histidine buffer, citric acid-citrate buffer and acetic acid-acetate buffer;

(3) sodium chloride at a concentration of about 140 mM;

wherein the liquid formulation has a pH of about 5.2 to about 6.8 (e.g., a pH of about 5.6 to about 6.8, such as a pH of about 5.6 to about 6.0, such as a pH of about 5.8); and, the liquid formulation does not contain a sugar and/or a sugar alcohol.

In certain preferred embodiments, the liquid formulations of the present invention may be stable at 5 ℃ ± 3 ℃ and may be stored for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, or at least about 2 years. In certain preferred embodiments, the liquid formulations of the present invention are stable at about 40 ℃ and can be stored for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, or at least about 2 years. In certain preferred embodiments, the liquid formulations of the present invention are stable at room temperature (e.g., about 25 ℃) and can be stored for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, or at least about 2 years.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, histidine-hydrochloride buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃ and stable for at least 12 months at 5 ℃ ± 3 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, histidine-hydrochloride buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.0. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, histidine-hydrochloride buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 7.0. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, histidine-acetate buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, acetic acid-sodium acetate buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, citrate-sodium citrate buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, histidine buffer as histidine alone at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃, for at least 6 months at 25 ℃, and for at least 36 months at 5 ℃ ± 3 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 140mM, histidine-histidine hydrochloride buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 28 days at 40 ℃, for at least 6 months at 25 ℃, and for at least 36 months at 5 ℃ ± 3 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60mg/ml, sodium chloride at a concentration of 140mM, histidine buffer as histidine alone at a concentration of 10mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.7. In such embodiments, the liquid formulation is stable for at least 14 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60mg/ml, sodium chloride at a concentration of 140mM, histidine-histidine hydrochloride buffer at a concentration of 10mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.7. In such embodiments, the liquid formulation is stable for at least 14 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60mg/ml, sodium chloride at a concentration of 140mM, histidine buffer as histidine alone at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. The liquid formulation is stable for at least 6 months at 5 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60mg/ml, sodium chloride at a concentration of 140mM, histidine-histidine hydrochloride buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. The liquid formulation is stable for at least 6 months at 5 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 90mg/ml, sodium chloride at a concentration of 140mM, histidine buffer as histidine alone at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 13 days at 40 ℃ and for at least 2 months at 25 ℃ ± 2 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 120mg/ml, sodium chloride at a concentration of 140mM, histidine-hydrochloride buffer at a concentration of 20mM, polysorbate 80 at a concentration of 0.02% w/v; and, the pH of the liquid formulation is 5.8. In such embodiments, the liquid formulation is stable for at least 13 days at 40 ℃ and for at least 2 months at 25 ℃ ± 2 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 100mM, citric acid-sodium citrate buffer at a concentration of 20mM, and the pH of the liquid formulation was 5.6. In such embodiments, the Tm value of the liquid formulation sample is greater than 65 ℃ and is stable for at least 3 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 100mM, citric acid-sodium citrate buffer at a concentration of 20mM, and the pH of the liquid formulation was 6.0. In such embodiments, the Tm value of the liquid formulation sample is greater than 65 ℃ and is stable for at least 3 days at 40 ℃.

In certain exemplary embodiments, the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20mg/ml, sodium chloride at a concentration of 100mM, citric acid-sodium citrate buffer at a concentration of 20mM, and the pH of the liquid formulation was 6.8. In such embodiments, the Tm value of the liquid formulation sample is greater than 65 ℃ and is stable for at least 3 days at 40 ℃.

In another preferred example, the specification of the liquid formulation is 150mg (10 ml)/bottle to 2000mg (10 ml)/bottle.

In another preferred embodiment, the specification of the liquid formulation is selected from the group consisting of: 200mg (10 ml)/bottle, 600mg (10 ml)/bottle, 900mg (10 ml)/bottle, 1200mg (10 ml)/bottle or 1500mg (10 ml)/bottle. The anti-PD-L1 antibody liquid preparation of the present invention can be prepared by combining the various components at predetermined concentrations using methods well known in the art. For example, the anti-PD-L1 antibody can be dialyzed into a solution containing the other components of the liquid formulation and adjusted to the desired concentration. The preparation was filter sterilized using a 0.22 μm pore size filter. The prepared preparation is packaged for convenient use. The packaging material may be a glass vial (e.g., a vial of penicillin), a metal alloy container, a pre-filled syringe, or a pen-type syringe.

The anti-PD-L1 antibody liquid formulations of the invention can be contained in any container suitable for storing drugs and other pharmaceutical compositions. For example, the liquid formulation of the present invention may be contained in a sealed and sterilized plastic container, metal alloy container or glass container having a certain volume, such as an ampoule, a vial or a syringe. Thus, in certain preferred embodiments, the liquid formulation of the present invention is contained in a glass vial (e.g., an ampoule or vial), a syringe, or a microinfusion set. In certain exemplary embodiments, the liquid formulations of the present invention are contained in a glass vial (e.g., an ampoule or vial).

Thus, in another aspect, the present invention also provides an article of manufacture comprising a container containing a liquid formulation of the present invention. In certain preferred embodiments, suitable containers include, for example, plastic containers, metal alloy containers, or glass containers, such as ampoules, vials, or syringes. In certain preferred embodiments, the article of manufacture of the invention comprises a container containing a liquid formulation of the invention, suitably a glass vial, a metal alloy container or a pre-filled syringe. In certain preferred embodiments, the article may further comprise a package insert printed with instructions for use.

The anti-PD-L1 antibody liquid preparations of the present invention can be administered to a subject by a parenteral route, such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or transdermal, mucosal, nasal, respiratory, and/or oral administration. In certain preferred embodiments, the liquid formulation is administered to a subject (e.g., a human) by intravenous injection or subcutaneous injection.

Therapeutic use of liquid formulations

The anti-PD-L1 antibody humoral preparation of the present invention can be used for the prevention and/or treatment of tumors or anemia by inhibiting and/or blocking intracellular signaling mediated by PD-1 binding to PD-L1.

Thus, in another aspect, the invention also relates to the use of a liquid formulation of the invention for the manufacture of a medicament for:

(a) prevention and/or treatment of tumors or anemia;

(b) adjuvant treatment of tumors or anemia;

(c) diagnosing a tumor or anemia;

(d) any combination of (a) - (c).

In certain embodiments, the liquid formulation of the invention is administered separately, in combination, simultaneously, or sequentially with an additional pharmaceutically active agent for the prevention and/or treatment and/or co-treatment of tumors or anemia. In certain embodiments, the additional pharmaceutically active agent is, for example, a chemotherapeutic drug. In certain preferred embodiments, the tumor is selected from the group consisting of breast cancer, lung cancer such as non-small cell lung cancer and squamous lung cancer, liver cancer, stomach cancer, intestinal cancer such as colon or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell cancer, cholangiocarcinoma, nasopharyngeal cancer, and head and neck squamous cell carcinoma, glioma, melanoma leukemia and lymphoma.

In certain preferred embodiments, the lymphoma is hodgkin's lymphoma or non-hodgkin's lymphoma. In certain preferred embodiments, the non-hodgkin's lymphoma is one or more of peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, NK/T cell lymphoma, and B cell non-hodgkin's lymphoma.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of pharmaceutical biochemistry, nucleic acid chemistry, immunological laboratories and the like used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, each heavy chain comprising a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region comprising three constant domains CH1, CH2 and ch3, each light chain comprising a light chain variable region (abbreviated herein as VL) and a light chain constant region, the light chain constant region comprising one constant domain cl.the VH and VL regions may also be subdivided into regions of high denaturation (termed Complementarity Determining Regions (CDRs)) interspersed with more conserved regions (termed Framework Regions (FR)) therebetween, each VH and VL being composed of 3 CDRs and 4 CDRs arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, the variable regions (VH and VL) of each heavy chain/light chain pair form antigen binding sites, respectively, the variable regions (VH and VL) form the antigen binding sites, and the heavy chain isotypes of IgG, IgA, IgG, kappa, and IgG, and kappa, IgG, and IgG, kappa, and IgG, kappa, and IgG, kappa, heavy chain isotypes, and IgG, and.

The term "antibody" includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; a human or non-human antibody; and (4) fully synthesizing the antibody. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise indicated, "antibody" as used herein should be understood to encompass an intact antibody molecule, i.e., an immunoglobulin IgG.

The term "anti-PD-L1 antibody" refers to an antibody that is capable of specifically binding to PD-L1. The term "specifically binds" refers to the formation of a complex of an antibody and an antigen that is relatively stable under physiological conditions. Specific binding can be achieved by about 1X 10^-9An equilibrium dissociation constant (K) of M or less_D) To characterize. Methods for determining whether two molecules specifically bind are well known in the art, such as bioluminescence interferometry or surface plasmon resonance techniques (e.g., Biacore). anti-PD-L1 antibodies (intact antibody molecules) suitable for use in the liquid formulations disclosed herein are presented in PCT patent publication No. WO2017/148424, which is incorporated herein by reference in its entirety. In certain preferred embodiments, the anti-PD-L1 antibodies described herein comprise heavy chain CDRs as shown in SEQ ID Nos. 1-3, respectively, and light chain CDRs as shown in SEQ ID Nos. 4-6, respectively. In certain preferred embodiments, the anti-PD-L1 antibody described herein has the heavy chain variable region shown as SEQ ID NO. 7 and the light chain variable region shown as SEQ ID NO. 8. In certain preferred embodiments, the anti-PD-L1 antibodies described herein have a heavy chain as shown in SEQ ID NO. 9 and a light chain as shown in SEQ ID NO. 10. The terminal Lys of the heavy chain is easily deleted, but this deletion does not affect the biological activity, see Dick, L.W. et al, Biotechnol.Bioeng., 100: 1132-1143.

As used herein, the term "programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed in vivo predominantly on previously activated T cells and binds to both ligands PD-L1 and PD-L2. The complete hPD-1 sequence can be found under GenBank accession No. U64863. Accordingly, the term "programmed death ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2) that down-regulates T cell activation and cytokine secretion when bound to PD-1. The term "PD-L1" as used herein includes human PD-L1(h PD-L1), variants, isoforms and species homologs of h PD-L1, and analogs having at least one epitope in common with h PD-L1. The complete h PD-L1 sequence can be found under GenBank accession No. Q9NZQ 7.

As used herein, the term "liquid formulation" means a combination of at least one active ingredient (e.g., an antibody, which is capable of exerting a biological activity in a subject) and at least one inactive ingredient, which when combined with the active ingredient, is suitable for administration to the subject. Typically, liquid formulations do not contain ingredients that are unacceptably toxic to the subject to which the formulation will be administered. Herein, the subject may comprise a mammal, preferably a human.

As used herein, the expression "stability" of an antibody means that an antibody substantially retains its physical and/or chemical stability and/or biological activity upon storage. The shelf life is generally selected based on the intended shelf life of the formulation. Analytical techniques for measuring antibody stability are well known in the art, and stability can be determined at a selected temperature for a selected time (e.g., antibody stability during storage at 40 ℃ for at least 28 days and testing, or antibody stability during storage at 5 ℃ ± 3 ℃ for at least 12 months and testing).

With respect to physical stability, an antibody in a formulation is considered to retain its physical stability if it exhibits substantially no evidence of significant aggregation, precipitation and/or denaturation as determined by visual inspection for color and/or clarity, or by UV light scattering or by size exclusion chromatography.

For chemical stability, assessment can be made by detecting and quantifying chemically altered forms of the antibody. Chemical changes may involve size changes (e.g., shearing), for example, as may be assessed by using size exclusion chromatography, SDS-PAGE and/or matrix-assisted laser-resolved ionization/time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., changes due to deamidation), which can be assessed, for example, by ion exchange chromatography.

With respect to biological activity, an antibody in one formulation retains its biological activity in the formulation if the antibody has the biological activity of its intended use. For example, an antibody in the formulation is considered to retain its biological activity (e.g., as determined by an antigen binding assay) if its biological activity is within about 70% to 130% (e.g., 80% to 130%) of the biological activity exhibited when the formulation was prepared.

As used herein, the term "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugated components. Examples of such buffers include acetate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. In certain preferred embodiments, the buffer suitable for use in the liquid formulations disclosed herein is selected from the group consisting of a citrate-citrate buffer, a histidine buffer, and an acetate-acetate buffer.

The term "histidine buffer" refers to a buffer comprising histidine, which histidine may be present alone or in the following forms: for example, histidine hydrochloride, histidine acetate, histidine phosphate or histidine sulfate. When histidine is not present alone, an amino acid buffer (e.g., histidine hydrochloride buffer) can be prepared by titrating L-histidine (free base, solid) with the corresponding acid (e.g., hydrochloric acid). When histidine is present in a separate form, subsequent manufacturing steps may incorporate pH adjusting agents as appropriate to obtain a liquid formulation of the appropriate pH range. In certain preferred embodiments, histidine in a histidine buffer suitable for use in the liquid formulations disclosed herein is present alone or as histidine hydrochloride or histidine acetate.

The term "citrate-citrate buffer" is a mixture comprising citric acid and citrate. In certain preferred embodiments, the citric acid-citrate buffer is a citric acid-sodium citrate buffer, i.e., comprises a mixture of citric acid and sodium citrate. A citric acid-citrate buffer (e.g., a citric acid-sodium citrate buffer) can be prepared by adding to citric acid a base (e.g., sodium hydroxide) necessary for salt formation.

The term "acetate-acetate buffer" is intended to include mixtures of acetic acid and acetate salts. In certain preferred embodiments, the acetate buffer is an acetate-sodium acetate buffer, i.e., a mixture comprising acetic acid and sodium acetate. An acetate-acetate buffer (e.g., an acetate-sodium acetate buffer) can be prepared by adding a base (e.g., sodium hydroxide) required for salt formation to acetic acid.

As used herein, the term "sugar" or "sugar alcohol" has the meaning commonly understood by those skilled in the art. The sugar comprises the general Composition (CH)₂O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, reducing sugars, non-reducing sugars, and the like, non-limiting examples of which include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran (dextran), and the like. Sugar alcohols can be prepared from the corresponding sugars by reducing the aldehyde or ketone groups on the sugar molecule to hydroxyl groups to obtain sugar alcohols; sugar alcohols have the general formula h (choh) nH, non-limiting examples of which herein include erythritol, threitol, arabitol, xylitol, sorbitol, mannitol, maltitol, lactitol, and the like.

The anti-PD-L1 antibody liquid formulation of the present invention does not contain sugars and/or sugar alcohols, and therefore the formulation may also be referred to herein as a sugarless antibody formulation. In certain preferred embodiments, the anti-PD-L1 antibody liquid formulations of the present invention do not contain a sugar and/or sugar alcohol selected from the group consisting of: sucrose, trehalose, glucose, mannitol and sorbitol. It is known in the art that in general, sugar and/or sugar alcohol (e.g., sucrose, trehalose, glucose, mannitol, sorbitol, etc.) are required to be added to an antibody preparation as a stabilizer for the antibody, and in addition, the sugar and/or sugar alcohol may also serve as an effective osmotic pressure regulator, which is capable of maintaining the stability of the preparation (e.g., isotonic, i.e., having substantially the same osmotic pressure as human blood).

As used herein, the term "osmolality" refers to a measure of the osmotic pressure of dissolved solute particles in an aqueous solution. The solute particles comprise two ions as well as non-ionized molecules. The osmotic pressure is expressed as the concentration (i.e., osmolality) of osmotically active particles dissolved in 1kg of solvent (i.e., water). Osmolality is expressed herein in units of milliosmoles per kilogram of water (mOsmol/kg). It is known to those skilled in the art that a desired level of osmolality can be achieved by the addition of one or more osmo-regulators, such as sugars and/or sugar alcohols.

As used herein, the term "isotonic" means that the formulation of interest has substantially the same osmotic pressure as human blood. In certain preferred embodiments, the isotonic formulation will generally have an osmotic pressure of about 260-330 mOsmol/kg. Isotonicity can be measured using, for example, a vapor pressure or freezing type osmometer.

As used herein, the term "mass to volume ratio" or "w/v" refers to the percentage weight (in grams) of a single component relative to the total volume (in milliliters) of a mixture containing that component. For example, 20mg of the component in a total volume of 100ml is 0.02% w/v.

As used herein, the term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. Herein, when "about" is used to describe a measurable value (e.g., concentration of a substance, time, temperature, etc.), a range of ± 5% of a given value is meant to be included, e.g., the expression "about 10 mM" means 9.5-10.5 mM.

Advantageous effects of the invention

In the preparation of antibody preparations, the addition of sugars and/or sugar alcohols is generally required in the art to maintain the stability of antibodies, however, the use of sugars and/or sugar alcohols has certain limitations for diabetic patients. Through a large number of experiments, the inventor of the application unexpectedly discovers that the sugar-free antibody preparation disclosed by the invention can maintain the osmotic pressure of the antibody preparation to obtain excellent physiological compatibility, can maintain the physical stability, chemical stability and biological activity of the preparation, and can tolerate severe conditions during storage and transportation, so that the sugar-free antibody preparation is particularly beneficial to long-term storage of medicines, can expand the clinical application range and has great clinical value.

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments.

Sequence information

Information on the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

The above CDRs may be obtained by techniques well known to those skilled in the art, such as analysis of the amino acid sequence of the following heavy chain variable region or the amino acid sequence of the light chain variable region according to the IMGT definition by the VBASE2 database.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Test materials and methods

Table 2: examples relate to reagents

Name of material	Manufacturer of the product
		Polysorbate 80	NANJING WELL CHEMICAL Co.,Ltd.
Histidine hydrochloride	Shanghai Xihe amino acids Co Ltd
		Histidine	Shanghai Xihe amino acids Co Ltd
Sodium chloride	Jiangsu diligence pharmaceutical Co Ltd
		Sucrose	Merck
Acetic acid	Hunan Er-Kang Pharmaceutical Co.,Ltd.
		Sodium acetate	Hunan Er-Kang Pharmaceutical Co.,Ltd.
Citric acid	Hunan Er-Kang Pharmaceutical Co.,Ltd.
		Citric acid sodium salt	Hunan Er-Kang Pharmaceutical Co.,Ltd.

The source of the anti-PD-L1 humanized monoclonal antibody in the following examples is described in PCT patent publication No. WO2017/148424, and has a heavy chain as shown in SEQ ID NO. 9 and a light chain as shown in SEQ ID NO. 10.

Detection method

(1) Protein concentration

Using a Solo-VPE variable path ultraviolet spectrophotometer, approximately 30. mu.l of the sample was pipetted into a cuvette and the sample protein concentration was determined by measuring the UV absorbance.

(2)Flowcam

The amount of particles in the sample was measured using Flowcam GWF-8JD, about 1ml of the sample was aspirated using a pipette tip, the tip containing the sample was placed over the sample port, and the amount of insoluble particles ≥ 10 μm and ≥ 25 μm in the sample was measured.

(3) Osmotic pressure

Placing 25 μ l sample in a sample tube, and placing the sample tube in a 819M freezing point osmometer

The osmolarity of the sample is measured under the probe.

(4) Molecular size exclusion chromatography (SEC-HPLC)

On a Waters e2695 HPLC column temperature was kept at room temperature, treated samples were injected using a TSKgel G3000SWXL column, mobile phase eluted for about 20min and size molecule distribution in samples determined by molecular size exclusion chromatography. The sample curves were derived from UV absorption at 280nm and the relative percent peak area of the total area was reported as the result.

(5) Ion exchange chromatography (IEC-HPLC)

The treated samples were injected on a Waters e2695 HPLC at 30 ℃ using a ProPac WCX-10 column, eluted with mobile phase and the distribution of the different charged ionic components in the samples was determined by ion exchange chromatography. The sample curves were derived from UV absorption at 280nm and the relative percent peak area of the total area was reported as the result.

(6) Capillary electrophoresis (CE-SDS)

And (2) adopting a sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, treating the sample, and quantitatively determining the purity of the recombinant monoclonal antibody product according to the molecular weight under the Non-reduced (NR) condition.

(7) Relative binding Activity (%)

An enzyme-linked immunosorbent assay (ELISA) is established to detect EC50 (half effective concentration) of the sample. The relative binding activity of the samples was calculated by comparing the EC50 of the samples to the reference, and the standard was initially set to 70-130% due to a detection deviation of ± 20% for this method.

Example 1

Prescription 1: the anti-PD-L1 humanized monoclonal antibody 20mg/ml, 100mM sodium chloride, 20mM citric acid-sodium citrate, pH 5.2.

Prescription 2: anti-PD-L1 humanized monoclonal antibody 20mg/ml, 100mM sodium chloride, 20mM citric acid-sodium citrate, pH 5.6.

Prescription 3: anti-PD-L1 humanized monoclonal antibody 20mg/ml, 100mM sodium chloride, 20mM citric acid-sodium citrate, pH 6.0.

Prescription 4: anti-PD-L1 humanized monoclonal antibody 20mg/ml, 100mM sodium chloride, 20mM citric acid-sodium citrate, pH 6.4.

Prescription 5: anti-PD-L1 humanized monoclonal antibody 20mg/ml, 100mM sodium chloride, 20mM citric acid-sodium citrate, pH 6.8.

The preparation method of the antibody preparation of the prescriptions 1-5 is as follows:

1. preparing a buffer solution: to water for injection, 100mM sodium chloride, 20mM citric acid, was added and adjusted to the desired pH value as prescribed above with sodium hydroxide.

2. The antibody stock solution (Sichuan Colubet biological medicine GmbH, batch No. 1) was ultrafiltered, dialyzed into the above buffer solution, and the antibody concentration was adjusted to 20 mg/ml.

3. And (3) sterilizing and filtering the prepared sample through a filter with the pore size of 0.22 mu m under the laminar flow condition, filling the sample into a penicillin bottle, and plugging and capping the penicillin bottle to obtain the sample.

The influence of the pH value of the preparation on the stability of the monoclonal antibody is examined by a DSF method, and the results show that the Tm values of samples with different pH values are all more than 65 ℃, which shows that the pH value has no significant influence on the thermal stability of the protein in the pH range of 5.6-6.8 (Table 3). The samples were placed at 40 ℃ for 3 days for accelerated experiments, and the SEC-HPLC results showed that the monomers were greater than 99.0%, no significant decrease, no oligomers were detected, the high polymers were less than 0.20%, no significant increase, and the stability of each sample was good (Table 4).

Table 3: tm values of samples with different pH values

Sample (I)	Prescription 1	Prescription 2	Prescription 3	Prescription 4	Prescription 5
						DSF Tm(℃)	65.31	66.25	66.25	66.33	66.40

Table 4: accelerated test results of samples with different pH values

Example 2

Prescription 6: the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine hydrochloride, polysorbate 80 with the mass-volume ratio of 0.02% and pH 5.8.

100ml of the antibody preparation of formula 6 was prepared as follows:

1. preparing a buffer solution: to water for injection, 140mM sodium chloride, 20mM histidine were added, adjusted to pH5.8 with hydrochloric acid.

2. The stock antibody solution (batch 1) was exchanged by ultrafiltration, dialyzed against the above buffer, adjusted to 20mg/ml antibody concentration, and added with a suitable amount of polysorbate 80 stock solution.

The stability of the sample of formula 6 at 40 ℃ for 28 days was examined, and the results are shown in tables 5 to 9, respectively.

Example 3

Prescription 7: the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine hydrochloride, polysorbate 80 with the mass-volume ratio of 0.02% and the pH value is 5.0.

The buffer pH was adjusted to 5.0 and the procedure was otherwise as in example 2.

The stability of the sample of formula 7 at 40 ℃ for 28 days was examined, and the results are shown in tables 5 to 9, respectively.

Example 4

Prescription 8: the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine hydrochloride, polysorbate 80 with the mass-volume ratio of 0.02% and the pH value is 7.0.

The buffer pH was adjusted to 7.0 and the procedure was otherwise as in example 2.

The stability of the sample of formula 8 at 40 ℃ for 28 days was examined and the results are shown in tables 5-9, respectively.

Example 5

Prescription 9: the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine acetate, 0.02% by mass/volume polysorbate 80, and pH 5.8.

To water for injection, 140mM sodium chloride and 20mM histidine were added, and the pH was adjusted to 5.8 with acetic acid, and the other preparation method was the same as that of example 2.

The stability of the sample of formula 9 at 40 ℃ for 28 days was examined, and the results are shown in tables 5 to 9, respectively.

Example 6

Prescription 10: the anti-PD-L1 humanized monoclonal antibody 20mg/ml, 140mM sodium chloride, 20mM acetic acid-sodium acetate, polysorbate 80 with the mass-volume ratio of 0.02%, and pH 5.8.

The same procedure as in example 2 was repeated except that 140mM sodium chloride and 20mM sodium acetate were added to water for injection and the pH was adjusted to 5.8 with acetic acid.

The stability of the sample of formula 10 at 40 ℃ for 28 days was examined and the results are shown in tables 5-9, respectively.

Example 7

Prescription 11: 20mg/ml of anti-PDL-1 humanized monoclonal antibody, 140mM of sodium chloride, 20mM of citric acid-sodium citrate, 0.02% of polysorbate 80 in mass-volume ratio, and pH 5.8.

The same procedure as in example 2 was repeated except that 140mM sodium chloride and 20mM sodium citrate were added to water for injection and the pH was adjusted to 5.8 with citric acid.

The stability of the sample of formula 11 at 40 ℃ for 28 days was examined, and the results are shown in tables 5 to 9, respectively.

Example 8

Prescription 12: the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine hydrochloride, 0.02% by mass/volume polysorbate 20, and pH 5.8.

In the formula 8, polysorbate 80 is replaced by polysorbate 20, and the other preparation methods are the same as those in example 2.

The stability of the sample of formula 12 at 40 ℃ for 28 days was examined, and some of the results are shown in tables 7-9.

Example 9

The samples of examples 2-8 above were tested for stability at 40 ℃ for 28 days.

9.1 osmotic pressure measurement

The results of the osmotic pressure measurements of examples 2-7 are shown in the following table. The results show that the samples of examples 2-7 were stable in osmotic pressure in the range of 280-320mOsmol/kg and good in stability when left at 40 ℃ for 28 days.

Table 5: osmotic pressure test results

9.2 insoluble particle detection

The samples of examples 2-7 were tested for insoluble particles by Flowcam and the results are shown in the table below. The results show that the number of particles larger than or equal to 10 mu m in each prescription sample is less than 1000, and the number of particles larger than or equal to 25 mu m in each prescription sample is less than 200, which indicates that the prescription 6-11 still has no obvious aggregation after being placed for 28 days at 40 ℃, and meets the requirement of medical use.

Table 6: flowcam test results

9.3 molecular size exclusion chromatography (SEC-HPLC) assay

The monomer content of the samples of examples 2-8 was determined by size exclusion chromatography and the results are shown in the table below. The results show that the monomer content of the samples of formulas 6-12 is more than 95%, and the samples are stable after being placed for 28 days at 40 ℃.

Table 7: SEC-HPLC detection results

9.4 ion exchange chromatography (IEC-HPLC) detection

The samples of examples 2-8 were tested for major peak content by ion exchange chromatography and the results are shown in the table below. The result shows that the main peak content of the prescription 6-12 is kept stable after being placed for 28 days at 40 ℃, and the quality requirement is met.

Table 8: IEC-HPLC test results

9.5 capillary electrophoresis (CE-SDS) detection

The samples of examples 2-8 were tested for major peak purity by capillary electrophoresis and the results are shown in the table below. The results show that after the sample is placed at 40 ℃ for 28 days, the main peak purity of the sample of the prescription 6-12 is high, the content is kept stable, and the quality requirement is met.

Table 9: non-reduced CE-SDS assay results

Purity of main peak (%)	0 day at 40 DEG C	14 days at 40 DEG C	28 days at 40 DEG C
				Example 2	96.3	95.9	95.0
Example 3	97.0	95.8	89.2
				Example 4	97.0	95.3	94.0
Example 5	96.8	95.9	93.4
				Example 6	97.0	95.7	93.1
Example 7	97.0	95.1	91.3
				Example 8	97.2	95.0	93.4

Example 10

The antibody preparation of formula 6 was prepared up to 10L by the following method:

1. preparing an ultrafiltration buffer solution: sodium chloride and histidine were added to water for injection, and the pH was adjusted to 5.8 with hydrochloric acid to prepare a solution containing 140mM sodium chloride and 20mM histidine-histidine hydrochloride.

2. Antibody stock solution (batch 2) was exchanged by ultrafiltration using Millipore ultrafiltration membrane cartridges, selecting the appropriate number of membranes depending on the loading and using a Thermo Pellicon ultrafiltration system. The protein was dialyzed into the above buffer, the antibody concentration was adjusted to 20mg/ml, and an appropriate amount of polysorbate 80 was added.

3. The prepared sample was subjected to sterile filtration under laminar flow conditions, and the filtration was performed using a Millipak series 0.22 μm filter (Millipore Co.). If desired, a prefiltration membrane Opticap filter can be placed in series before the sterilizing filter to increase filter loading. The sterilizing filter is connected with a peristaltic pump by a silica gel tube in a sterile environment (super clean bench), and is sterilized and filtered by a filter with the pore diameter of 0.22 mu m. And collecting the filtered sample solution, filling the sample solution into penicillin bottles respectively, and plugging and capping the penicillin bottles to obtain the sample.

The long-term stability study at 5 ℃. + -. 3 ℃ for 12 months was carried out, and the investigation items were protein concentration, SEC-HPLC, CE-SDS, and IEC-HPLC, and the results are shown in Table 10. The result shows that the anti-PD-L1 humanized monoclonal antibody liquid preparation with the prescription can be produced in an enlarged mode, the quality of the liquid preparation can still reach the same quality as that of a small-scale preparation through the enlarged production, the antibody concentration is stable, the purity is kept higher, the stability is good, and the liquid preparation can be stably stored for at least 12 months under the temperature condition of 5 +/-3 ℃.

Table 10: long term stability test data for prescription 6

In addition, the freeze-thaw stability of formulation 6 was studied at-20 ℃ for 48 hours for OD405, SEC-HPLC and IEC-HPLC, and the results are shown in Table 11. The results show that the anti-PD-L1 humanized monoclonal antibody liquid formulation of this formulation has good stability for 4 cycles of freeze-thaw, and can withstand the rigors of the formulation during storage and transportation.

Table 11: freeze-thaw stability test data for prescription 6

Another batch of the same formulation of antibody stock (batch No. 3) was prepared in the same manner as described above in this example. Long-term stability studies at 5 + -3 deg.C and accelerated stability studies at 25 + -2 deg.C were performed for formulation 6, with the study items being appearance, protein concentration, pH, osmotic pressure, insoluble microparticles, SEC-HPLC, CE-SDS, IEC-HPLC, binding activity. The results are as follows:

table 12: 5 +/-3 ℃ long-term stability research and detection data of prescription 6

The prescription 6 is placed for 36 months at the temperature of 5 ℃, the appearance is clear and transparent, the protein content, the pH value, the molar osmotic pressure, the CE-SDS reduction and non-reduction, the SEC-HPLC, the IEC-HPLC and the relative binding activity are not obviously changed, and the stability is better.

Table 13: 25 +/-2 ℃ accelerated stability research and detection data of prescription 6

The prescription 6 is placed for 6 months under the accelerated condition of 25 +/-2 ℃, the appearance is clear and transparent, the protein content, the pH, the osmotic pressure and the relative binding activity are not obviously changed, the CE-SDS reduction and non-reduction, the SEC-HPLC and IEC-HPLC purities are slightly reduced, the protein stability is good, and the quality requirement is met.

Example 11

Prescription 16: the anti-PD-L1 humanized monoclonal antibody is 60mg/ml, 10mM histidine-histidine hydrochloride, 140mM sodium chloride, polysorbate 80 with the mass-volume ratio of 0.02% and the pH value is 5.7. The antibody concentration in the buffer was 60mg/ml, and the other preparation methods were the same as in example 2.

The stability test results at 40 ℃ are shown in table 14, and the appearance of the sample of formula 16 is a clear transparent liquid with slight opalescence; also, OD405 was stable over time at 40 ℃ indicating stability of the formulation.

Table 14: stability test results for prescription 16

Example 12

Prescription 17: the anti-PD-L1 humanized monoclonal antibody 60mg/ml, 20mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% w/v polysorbate 80, pH5.8, was prepared in the same manner as in example 2.

Prescription 18: anti-PD-L1 humanized monoclonal antibody 90mg/ml, 20mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% w/v polysorbate 80, pH5.8, and its preparation method is the same as example 2.

Prescription 19: anti-PD-L1 humanized monoclonal antibody 120mg/ml, 20mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% w/v polysorbate 80, pH5.8, and its preparation method is the same as example 2.

The viscosity of recipe 6, recipe 17, recipe 18 and recipe 19 and the viscosity change at different temperatures were examined, respectively. The results are shown in Table 15, where the viscosity of formula 6 is lower at low concentrations and the viscosities of formulas 17, 18 and 19 gradually increase with increasing concentration. Recipe 6 and recipe 17 were left at 5 ℃ for 6 months with no significant change in viscosity compared to the initial; recipe 18 and recipe 19, which were left at 5 ℃ and 25 ℃ for 3 months, showed no significant change in viscosity compared to the initial viscosity. The high-concentration preparation is generally high in viscosity, and the viscosity of the preparation is controllable and stable at high concentration, so that the quality requirement is met.

Table 15: viscosity of recipes 6 and 17-19 and viscosity values at different temperatures

NT represents not detected.

Example 13

Stability of formula 18 and formula 19 was examined under shaking, 40 ℃, 25 ℃ and 5 ℃ conditions, respectively. As shown in Table 16, after 2 hours of continuous shaking at 1000rpm, the recipe 18 and the recipe 19 were not significantly changed and the stability was good. The preparation can be placed for 2 months at 5 ℃, all physical and chemical examination items of the prescription 19 basically remain unchanged, and the stability is good, which indicates that the finished product of the prescription 19 preparation can be placed for at least 2 months at 5 +/-3 ℃. When the preparation is placed for 2 months at the accelerated temperature of 25 ℃, the IEC main peak of the prescription 18 and the IEC main peak of the prescription 19 are slightly reduced, and the rest items are basically unchanged, which shows that the finished preparation of the prescription 18 and the prescription 19 can be placed for at least 2 months at the temperature of 25 +/-2 ℃. After the preparation is placed for 13 days at the temperature of 40 ℃, the IEC main peak and the CE-SDS non-reduction main peak of the prescription 18 and the prescription 19 are slightly reduced, other inspection items are not obviously changed, and the quality requirements are met, which indicates that the finished products of the preparation of the prescription 18 and the prescription 19 can be placed for at least 13 days at the temperature of 40 ℃. The preparation overcomes the problems of stability, viscosity, osmotic pressure and turbidity, and is beneficial to long-term storage and clinical medication of the medicine.

Table 16: the stability test results of the formula 18 and the formula 19 under the conditions of oscillation, 40 ℃, 25 ℃ and 5 ℃.

NT represents not detected.

Comparative example 1

Prescription 13: the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sucrose, 20mM histidine-histidine hydrochloride, 0.02% by mass/volume polysorbate 80, and pH 5.8. 140mM sodium chloride in the buffer was replaced with 140mM sucrose and the procedure was otherwise as in example 2.

Prescription 14: the anti-PD-L1 humanized monoclonal antibody 20mg/ml, 105mM sucrose, 35mM sodium chloride, 20mM histidine-histidine hydrochloride, polysorbate 80 with a mass-volume ratio of 0.02%, and pH 5.8. The buffer was replaced with 105mM sucrose and 35mM sodium chloride at 140mM sodium chloride, and the procedure was otherwise the same as in example 2.

Prescription 15: the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 70mM sucrose, 70mM sodium chloride, 20mM histidine-histidine hydrochloride, 0.02% by mass/volume polysorbate 80, and pH 5.8. 140mM sodium chloride in the buffer was replaced with 70mM sodium chloride and 70mM sucrose, and the other preparation was performed in the same manner as in example 2.

Through detection, the osmotic pressure of the prescription 13-15 is 199mOsmol/kg, 232mOsmol/kg and 258mOsmol/kg respectively, which are all lower than the osmotic pressure of the blood plasma of a normal human body, the antibody preparation is a hypotonic solution, and intravenous administration has the risk of causing hemolysis and does not meet the requirement of clinical medication.

Summary all formulations and their respective ingredients referred to in the examples of the present invention are summarized in table 17.

Table 17 respective part numbers and respective compositions thereof

Firstly, the results of Tm value measurement and stability measurement experiments at 40 ℃ of the antibody preparations of formulas 1 to 5 show that the antibody preparations of formulas 1 to 5 have good stability. This also indicates that the PD-L1 antibodies of the invention all have a better stability when the pH is in the range of 5.6-6.8.

Subsequently, the antibody preparation with the prescription 6-12 is subjected to osmotic pressure monitoring, insoluble particle monitoring, SEC-HPLC detection, IEC-HPLC detection and CE-SDS detection for 28 days at 40 ℃, and the results show that the antibody preparation with the prescription 6-12 has good stability and no obvious aggregation, and meets the pharmaceutical requirements. Also, the results show that the stability of the antibody of the present invention is optimal when the pH is 5.8, and that the stability effect is relatively superior to that of the other buffer pair when the buffer pair in the formulation is histidine-histidine hydrochloride.

Therefore, the inventors further examined and analyzed the antibody preparation of formula 6, which is relatively more effective. Specifically, the stability of the protein at 5 ℃ +/-3 ℃ for 12 months for a long time and the stability of the protein at 5 ℃ for 36 months for a long time are analyzed, and the protein concentration, SEC-HPLC, CE-SDS, IEC-HPLC, binding activity and the like are respectively tested; furthermore, the freeze-thaw stability at-20 ℃ for 48 hours was analyzed, and OD405, SEC-HPLC and IEC-HPLC were respectively tested. The result shows that the antibody preparation of the prescription 6 can be stably stored for at least 12 months at 5 +/-3 ℃, and the product quality same as that of a small-scale preparation can be still achieved through scale-up production; moreover, after repeated freeze thawing for 4 cycle periods, the stability is good, and the severe conditions of the preparation during storage and transportation can be tolerated; in addition, after being placed for 36 months at 5 ℃, the appearance is clear and transparent, the protein content, the pH value, the molar osmotic pressure, the reduction and non-reduction of CE-SDS, SEC-HPLC, IEC-HPLC and relative binding activity are not obviously changed, and the stability is better.

Further, the present inventors tried the antibody preparation under the condition of high antibody concentration-recipes 16 to 19, and tried the stability of the antibody preparation in the case of the PD-L1 antibody concentrations of 60mg/ml, 90mg/ml, and 120mg/ml, respectively. The results show that the antibody preparation of formula 16 has a clear transparent liquid with slight opalescence and stable OD405 at 40 ℃; under different temperature conditions, the viscosity of the antibody preparations of formulas 17-19 did not change significantly over time; the antibody preparations of the prescriptions 18 and 19 have good stability after continuous shaking for 2h under the condition of 1000rpm, can be placed for 2 months at 5 ℃ and 25 ℃ and can be placed for 13 days at 40 ℃. Therefore, in the invention, the antibody preparation under high concentration antibody concentration can overcome the problems of stability, viscosity, osmotic pressure and turbidity, and is beneficial to long-term storage and clinical medication of the medicine.

In addition, the inventors performed a screen-prescription 13-15 (comparative) on the concentration of NaCl in the antibody preparation. The results show that when the NaCl concentration is respectively 0mM, 35mM and 70mM, the osmotic pressure is respectively 199mOsmol/kg, 232mOsmol/kg and 258mOsmol/kg, which are all lower than the normal human plasma osmotic pressure, and the intravenous administration has the risk of hemolysis, thereby not meeting the clinical medication requirement.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Sequence listing

<110> Sichuan Kolun Bordete biomedical corporation

<120> an anti-PD-L1 antibody preparation

<130>IDC190429

<150>CN201811441890.4

<151>2018-11-29

<160>10

<170>PatentIn version 3.5

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<213> Artificial Sequence (Artificial Sequence)

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Gln Ser Ile Gly Thr Asn

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<213> Artificial Sequence (Artificial Sequence)

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Tyr Ala Ser

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<213> Artificial Sequence (Artificial Sequence)

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Gly Val Ile Trp Thr Gly Gly Ala Thr Asn Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn GlnVal Ser Leu

65 70 75 80

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Ser Asn Tyr Arg Tyr Asp Glu Pro Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>8

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Glu Ile Val Leu Thr Gln Ser Pro Asp Thr Leu Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Leu Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>9

<211>450

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>9

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr

20 25 30

Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Thr Gly Gly Ala Thr Asn Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ser Arg Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Asp Ser Asn Tyr Arg Tyr Asp Glu Pro Phe Thr Tyr Trp Gly Gln

100105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210>10

<211>214

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>10

Glu Ile Val Leu Thr Gln Ser Pro Asp Thr Leu Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Leu Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Trp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. A liquid formulation comprising:

(1) an anti-PD-L1 antibody at a concentration of about 10mg/ml to about 150 mg/ml; wherein the anti-PD-L1 antibody comprises heavy chain CDRs as shown in SEQ ID NO:1-3, respectively, and light chain CDRs as shown in SEQ ID NO:4-6, respectively;

(2) a buffer at a concentration of about 5mM to about 40 mM;

(3) sodium chloride at a concentration of about 80mM to about 160 mM;

wherein the liquid formulation has a pH of about 5.0 to about 7.0 and the liquid formulation is absent added sugar and/or sugar alcohol;

also, liquid formulations have an osmotic pressure comparable to human blood.

2. The liquid formulation of claim 1, wherein the anti-PD-L1 antibody has a heavy chain variable region as set forth in SEQ ID NO. 7 and a light chain variable region as set forth in SEQ ID NO. 8;

preferably, the anti-PD-L1 antibody has a heavy chain as shown in SEQ ID NO. 9 and a light chain as shown in SEQ ID NO. 10.

3. The liquid formulation of claim 1 or 2, wherein the anti-PD-L1 antibody is at a concentration of about 15mg/ml to about 25mg/ml, about 50mg/ml to about 70mg/ml, or about 80mg/ml to about 150 mg/ml;

preferably, the concentration of the anti-PD-L1 antibody is about 20mg/ml, about 60mg/ml, about 90mg/ml or about 120 mg/ml.

4. The liquid formulation of any one of claims 1-3, wherein the liquid formulation does not contain a sugar and/or sugar alcohol selected from the group consisting of: sucrose, glucose, trehalose, mannitol and sorbitol.

5. The liquid formulation of any one of claims 1-4, wherein the sodium chloride is at a concentration of about 100mM to about 160mM, such as about 110mM, about 120mM, about 130mM, about 140mM, about 150mM, about 155 mM;

preferably, the concentration of sodium chloride is about 140 mM.

6. The liquid formulation of any one of claims 1-5, wherein the buffer is selected from the group consisting of: a citrate-citrate buffer, a histidine buffer, or an acetate-acetate buffer; histidine in the histidine buffer exists alone or in the form of histidine-hydrochloride or histidine-acetate.

7. The liquid formulation of any one of claims 1-6, wherein the buffer is at a concentration of about 10mM to about 20 mM.

8. The liquid formulation of any one of claims 1-7, wherein the liquid formulation has a pH of about 5.2 to about 7.0;

preferably, the liquid formulation has a pH of about 5.6 to about 6.8;

preferably, the liquid formulation has a pH of about 5.6 to about 6.0; preferably, the liquid formulation has a pH of 5.8 ± 0.1.

9. The liquid formulation of any one of claims 1-8, wherein the osmolality comparable to human blood is an osmolality of about 280 to about 380 mOsmol/kg;

preferably, the liquid formulation has an osmolality of about 280mOsmol/kg to about 360 mOsmol/kg.

10. The liquid formulation of any one of claims 1-9, wherein the liquid formulation further comprises a surfactant; the surfactant is polysorbate 80, polysorbate 20 or poloxamer 188;

wherein the concentration of the surfactant is from about 0.01% w/v to about 0.02% w/v.

Preferably, the surfactant is polysorbate 80 at a concentration of about 0.02% w/v.

11. The liquid formulation of claims 1-10, the ingredients of the liquid formulation being selected from the group consisting of:

(a) the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine hydrochloride, polysorbate 80 with the mass-volume ratio of 0.02% and the pH value is 5.8;

(b) the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine hydrochloride, polysorbate 80 with the mass-volume ratio of 0.02% and the pH value is 5.0;

(c) the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine acetate, polysorbate 80 with the mass-volume ratio of 0.02%, and the pH value is 5.8;

(d) the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM acetic acid-sodium acetate, polysorbate 80 with the mass-volume ratio of 0.02% and the pH value is 5.8;

(e) 20mg/ml of anti-PDL-1 humanized monoclonal antibody, 140mM of sodium chloride, 20mM of citric acid-sodium citrate, 0.02% of polysorbate 80 in mass-volume ratio, and pH 5.8;

(f) the anti-PD-L1 humanized monoclonal antibody is 20mg/ml, 140mM sodium chloride, 20mM histidine-histidine hydrochloride, 0.02% by mass/volume polysorbate 20, and pH 5.8;

(g) 60mg/ml of anti-PD-L1 humanized monoclonal antibody, 10mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% by mass/volume polysorbate 80, pH 5.7;

(h) anti-PD-L1 humanized monoclonal antibody 60mg/ml, 20mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% w/v polysorbate 80, pH 5.8;

(i) anti-PD-L1 humanized monoclonal antibody 90mg/ml, 20mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% w/v polysorbate 80, pH 5.8;

(j) anti-PD-L1 humanized monoclonal antibody 120mg/ml, 20mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% w/v polysorbate 80, pH 5.8;

(k) anti-PD-L1 humanized monoclonal antibody 20mg/ml, 100mM sodium chloride, 20mM citric acid-sodium citrate, pH5.6;

(l) anti-PD-L1 humanized monoclonal antibody 20mg/ml, 20mM histidine-histidine hydrochloride, 140mM sodium chloride, 0.02% polysorbate 80, pH 7.0.

12. An article of manufacture comprising a container containing the liquid formulation of any one of claims 1-11.

13. The article of claim 12, wherein the container is a glass bottle, a metal alloy container, or a pre-filled syringe.

14. Use of a liquid formulation according to any one of claims 1 to 11 or a preparation according to claim 12 in the manufacture of a medicament for:

(a) prevention and/or treatment of tumors or anemia;

(b) adjuvant treatment of tumors or anemia;

(c) diagnosing a tumor or anemia;

(d) any combination of (a) - (c).

15. The use according to claim 14, wherein the medicament is administered separately, in combination, simultaneously, or sequentially with an additional pharmaceutically active agent; wherein the additional pharmaceutically active agent is a chemotherapeutic agent.

16. Use according to claim 14, characterized in that the tumour is selected from the group consisting of breast cancer, lung cancer such as non-small cell lung cancer or lung squamous cancer, liver cancer, stomach cancer, intestinal cancer such as colon or rectal cancer, oesophageal cancer, ovarian cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell cancer, bile duct cancer, nasopharyngeal cancer, head and neck squamous cancer, glioma, melanoma leukaemia and lymphoma.

17. The use of claim 16, wherein said lymphoma is hodgkin's lymphoma or non-hodgkin's lymphoma; more preferably, the non-hodgkin's lymphoma is one or more of peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma, NK/T cell lymphoma and B cell non-hodgkin's lymphoma.